This project will examine the role of determinants encoded by highly transmissible plasmid pCF10 of Enterococcus faecalis, and how these genes mediate plasmid transfer and persistence of enterococci in the mammalian gastrointestinal tract. E. faecalis is a leading cause of hospital-acquired infections whose treatment is impeded by the inherent and acquired antibiotic resistance of the organism, and by its ability to transition between commensal and pathogenic lifestyles. It is well understood that antibiotic treatment or impairment of normal host defenses can result in overgrowth of enterococci in the intestine. However, we know very little about the inherent traits that allow the organism to establish in a healthy intestine, or how antibiotic-resistance plasmids like pCF10 might impact competitive fitness of their bacterial host in the GI tract independent of antibiotic resistance. We will study the interactions between pCF10-carrying donor strains and plasmid-free recipients in germ-free mice, and in humanized animals carrying a human-derived gut microbial community. We will use powerful new screens to identify genetic determinants of competitive fitness and plasmid transfer in the GI tract, and begin to study their functions and mechanisms of action. We will use high-resolution microscopic techniques to examine the behavior of the organisms in vivo, and the expression of genes of interest. The results of this research will include the following: 1) We will determine the inherent frequencies of pCF10 transfer in various regions of the GI tract in the absence of antibiotic selection. 2) We will identify genes on both the plasmid and chromosome that impact transfer and competitive fitness. 3) We will use mathematical modeling and laboratory studies of regulatory mechanisms to correlate the functions of genetic control circuits to the enterococcal behavior in the natural environment. 4) In the longer term, we may identify new ecologically based strategies, which could reduce enterococcal infections or the spread of antibiotic resistance.